Professional Documents
Culture Documents
A THESIS SUBMITTED TO
THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES
OF
THE MIDDLE EAST TECHNICAL UNIVERSITY
BY
CEREN OKTAR
SEPTEMBER 2003
Approval of the Graduate School of Natural and Applied Sciences
I certify that this thesis satisfies all the requirements as a thesis for the degree
of Master of Science.
This is to certify that we have read this thesis and that in our opinion it is fully
adequate, in scope and quality, as a thesis and for the degree of Master of
Science.
ii
ABSTRACT
Oktar, Ceren
In this study, firstly the effects of aspartic acid group amino acids -which were
pHV1434::subC gene. All aspartic acid group amino acids except threonine
inhibited SAP activity when CAA≥ 2.5 mM. The highest SAP activities with
iii
found to be 1.89-, 1.87-, 1.61-, 1.48-, 1.4-, and 1.4-fold higher than the
acid was CAA=0.25 mM. The product and by-product distributions in defined and
complex media in SAP production were also analyzed and compared in order to
highest SAP activity in complex medium was found to be 3–fold higher than
defined medium activity, while, specific SAP production rate was 1.2- fold higher.
The highest cell concentration in complex medium (CX= 14.3 g/dm-3) was 8.1-
fold higher than that obtained in defined medium (CX= 1.75 g/dm-3). In both
complex medium there was also succinic acid in the extracellular medium
indicating that the operation of TCA cycle was insufficient. In both media serine,
valine and glycine were observed neither in the extracellular nor in the
intracellular media indicating that the synthesis of these amino acids can be a
secondary rate limiting step. In defined medium asparagine was present neither
in the cell nor in fermentation broth whereas, methionine was observed in the
cell in high amounts, probably due to the lower flux values towards asparagine.
iv
ÖZ
KARŞILAŞTIRMALI ANALİZİ
Oktar, Ceren
bütün aspartik asit grubu amino asitler CAA≥ 2.5 mM iken SAP aktivitesini inhibe
etmiştir. Herbir amino asit derişimi, CAA= 0.25 mM iken; asparajin, aspartik asit,
v
lözin, triyonin, izolözin ve metiyoninin için elde edilen en yüksek SAP aktiviteleri
sırasıyla referans ortam aktivitesinin 1.89, 1.87, 1.61, 1.48, 1.4 ve 1.4 katı
dm-3) 8.1-katı olarak bulunmuştur. Her iki ortamda da okzalik asit hem hücre
içinde hem de hücre dışında gözlenmiştir. Kompleks ortamda ayrıca süksinik asit
düşük oluşundan kaynaklanmaktadır. Sonuç olarak aspartik asit grup amino asit
potansiyeline sahiptir.
vi
To The Memory of My Grandfather and Grandmother,
vii
ACKNOWLEDGEMENTS
Prof. Pınar Çalık for introducing me to this research subject, for her invaluable
I would also like to express my gratitude to Prof. H. Tunçer Özdamar for being
an accessible source of support and guidance. And I wish to thank all the
I also wish to give my special thanks to all my friends in our research group in
valuable help during long laboratory nights in METU. I also wish to thank my
I would like to thank TUBITAK-BAYG for Münir Birsel Scholarship, TUBITAK for
the financial support provided through projects MISAG-176 and SPO through
Grant 2001K121030.
Finally, I am grateful to my family for all the wonderful and invaluable things
they have given me; support, understanding, love, and above all, a home.
viii
TABLE OF CONTENTS
ÖZ.......................................................................................................... v
DEDICATION...........................................................................................vii
NOMENCLATURE.....................................................................................xvi
CHAPTER
1 INTRODUCTION ................................................................................ 1
2 LITERATURE SURVEY......................................................................... 9
2.1 Enzymes .................................................................................... 9
2.2 Proteolytic Enzymes................................................................... 11
2.2.1 Serine Proteases .............................................................. 11
2.2.1.1 Serine Alkaline Protease (SAP) ................................ 12
2.3 Bioprocess Parameters for Serine Alkaline Protease Production......... 13
2.3.1 Microorganism ................................................................. 13
2.2.1.1 The Genus Bacillus ................................................ 13
2.3.2 Medium Design ................................................................ 14
2.3.2.1 Cell composition.................................................... 15
2.3.2.2 Types of Media...................................................... 18
2.3.3 Bioreactor Operation Parameters ........................................ 23
2.3.3.1 Temperature and pH.............................................. 23
2.3.2.1 Oxygen Transfer ................................................... 25
2.4 Intracellular Biochemical Reactions .............................................. 25
2.4.1 Synthesis of Aspartic Acid Group Amino Acids ....................... 27
ix
3 MATERIALS AND METHOD ................................................................ 29
3.1 Microorganism .......................................................................... 29
3.1.1 Microorganism Storage...................................................... 29
3.2 Solid Medium ............................................................................ 29
3.3 Precultivation Medium ................................................................ 29
3.4 Production Medium .................................................................... 31
3.5 Analysis .................................................................................. 32
3.5.1 Cell Concentration ........................................................... 33
3.5.2 Serine Alkaline Protease (SAP) Activity ................................ 33
3.5.3 Reduced Sugar Concentration by DNS Method ...................... 33
3.5.4 Determination of Amino Acid Concentrations ........................ 34
3.5.5 Determination of Organic Acid Concentrations ...................... 35
3.5.6 Determination of Intracellular Metabolite Concentrations ........ 35
3.5.6.1 DNA Concentration by Diphenylamine Reaction......... 36
3.5.6.2 RNA Concentration by Diphenylamine Reaction ......... 37
3.5.6.3 Determination of Intracellular Amino Acid and Organic
Acid Concentrations .............................................. 38
x
4.7.8 Variations in the Intracellular Amino Acid and Organic Acid
Concentrations in Complex Medium .................................. 60
4.7.9 Variations in the DNA and RNA Concentrations in Defined and
Complex Media .............................................................. 64
4.7.10 Comparison of Product and By-Product Distributions in Defined
and Complex Media ........................................................ 64
REFERENCES ......................................................................................... 73
APPENDICES .......................................................................................... 78
xi
LIST OF TABLES
3.3 Composition of Reference Defined Medium for Recombinant Bacillus sp. ... 31
Medium ........................................................................................... 51
Medium ........................................................................................... 52
Medium ........................................................................................... 54
Medium ........................................................................................... 54
xii
4.5 Variations in the Extracellular Amino Acid Concentrations in Complex
Medium ........................................................................................... 62
Medium ........................................................................................... 62
Medium ........................................................................................... 63
Medium ........................................................................................... 63
xiii
LIST OF FIGURES
4.7 Variations of Glucose and Cell Concentrations in Defined Medium with the
4.9 Variations in SAP Activity and Specific SAP Activity in Defined Medium
4.10 Variations in Specific SAP Production Rate in Defined Medium with the
xiv
4.13 Variations in SAP Activity and Specific SAP Activity in Complex Medium
4.14 Variations in Specific SAP Production Rate in Complex Medium with the
xv
LIST OF SYMBOLS AND ABBREVIATIONS
Ac Acetate
Ala L-Alanine
Arg L-Arginine
Asn L-Asparagine
Asp L-Aspartate
CaP Carbamoyl-phosphate
Chor Chorismate
CO2 Carbondioxide
xvi
Cys L-Cysteine
Fum Fumarate
Gln L-Glutamine
Glu L-Glutamate
Gly L-Glycine
His L-Histidine
Ile L-Isoleucine
αKG α-ketoglutarate
Kval Ketovaline
Leu L-Leucine
Lys L-Lysine
mDAP meso-Diaminopimelate
Met L-Methionine
OA Oxalacetate
Orn Ornithine
PEP Phosphoenolpyruvate
Phe L-Phenylalanine
Pi Inorganic ortophosphate
Pro L-Proline
Pyr Pyruvate
xvii
rP Specific serine alkaline protease production rate, U gX-1h-1
Ser L-Serine
t Residence time, h
TE Tris EDTA
THF Tetrahydrofolate
Thr L-Threonine
Trp L-Tyrptophan
Tyr L-Tyrosine
UDPNAG UDP-N-Acetyl-glucosamine
Val L-Valine
xviii
CHAPTER 1
INTRODUCTION
Enzymes are a major resource utilized by the food, chemical, and allied
time,
Table 1.1 summarizes the sources and application areas of some industrially
bacteria, fungi, animal and plant cells in order to be used in detergent, food
1
Table 1.1 Some examples of industrially important enzymes, their sources and
application areas
In 1998, the world-wide enzyme sales amounted to over $1.5 billion (OEDC,
1998). Table 1.2 summarizes the major producers of industrial enzymes. The
Gist-brocades (Netherlands) 20
Hansen (Denmark) 5
Sanofi (France) 5
Others 15
2
Most commercial enzymes are produced by microorganisms belonging to the
Bacillus and Aspergillus genera. The majority of the Bacillus species are harmless
and are well known for their ability to excrete enzymes such as amylases and
Serine alkaline proteases (SAP) are one of the most important group of industrial
enzymes that are widely used in detergent, leather and meat industries. They
account for approximately 35% of the microbial enzyme sales. Serine alkaline
proteases catalyze the hydrolysis of peptide bonds and have serine, histidine and
aspartic acid residues in the active site. They are most active around pH 10 and
must be taken into account in order to have high product yield and selectivity.
These are; (1) selection of microorganism, (2) medium design and (3) bioreactor
to obtain the desired product. The microorganism that is to be used should give
adequate yields, be able to secrete large amounts of protein and should not
produce toxins or any other undesired products. Serine alkaline proteases, like
Bacillus species since they are able to secrete large number of extracellular
enzymes (Priest, 1977). In bioprocesses carbon and energy sources and their
concentrations are indeed important as they are tools for bioprocess medium
design (Çalık et al., 2001). Culture medium supplies the microorganism with all
synthesizing all of their cellular constituents from carbon and nitrogen sources.
amino acids, trace elements, vitamins, etc.). The culture conditions that promote
3
production of enzymes like proteases are frequently significantly different from
the culture conditions promoting cell growth (Moon and Parulekar, 1991).
growth and product formation. There are three types of media; defined, semi-
the attainable enzyme activity and cell yields are much higher than that of
bioprocesses require high oxygen transfer rate conditions while others require
controlled oxygen transfer rate conditions (Çalık et al., 1999). Product yield and
of reactions take place in the microbioreactor that are coupled with the medium
a very large number of chemical reactions that occur inside individual cells and
intracellular substrates into biomass and metabolic products and then the
metabolic products are excreted back into the extracellular medium. SAP
synthesis depends on good coupling of supply and demand of the amino acids in
the cell, the syntesis of which are dependent on the intracellular concentrations
activities.
4
In the literature there have been various publications related with protease
poorly utilized carbon and nitrogen sources; then in 1982 they studied the
and fed-batch cultures of Bacillus firmus. Wright et al. (1992) investigated the
glucose or fructose for Bacillus brevis. Hübner et al. (1993) used both semi-
From the same research group van Putten et al. (1996) used the same medium
with Hübner et al. (1993) and tested various control strategies on pH and
using Bacillus licheniformis. Çalık et al. (1998) studied the effects of oxygen
defined medium where citric acid was the sole carbon source. Çalık et al. (1999),
5
wild type Bacillus licheniformis in relation to the physiology of the bacilli in a
article in 2000 Çalık et al. investigated the effects of oxygen transfer on product
conditions and with the provided constant oxygen transfer conditions they
bioprocess. Christiansen et al. (2002) investigated the uptake of the amino acids
amino acids, normally present in proteins using fully labeled glucose in batch
cultivation and they also analyzed the structure of the metabolic network of
B.clausii and estimated the metabolic fluxes in batch and continuous culture on a
minimal medium. Çalık et al. (2002a) used glucose as the sole carbon source in
of the findings of the previous study, the same research group investigated the
pH=6.8 and 7.25 on product and by-product formations as well as the oxygen
whereupon the rate limiting step of the bioprocess. Although the activity
obtained with citrate (Çalık et al., 2000) was higher than that of glucose (Çalık
et al., 2002) the enzyme was not able to maintain its high activity; therefore,
Çalık et al. (2002) used glucose as the carbon source. Following this study, in
order to increase SAP production Çalık et al. (2003a) cloned SAP encoding gene
6
source glucose under well-defined bioreactor operation conditions. Thereafter,
species the highest SAP production was obtained with recombinant B.subtilis
carrying pHV1431::subC. In the same study they also investigated the effects of
oxygen transfer in SAP production process in larger scale together with the
oxygen transfer parameters. In the literature there are no studies related with
the influence of aspartic acid amino acids on SAP production, which were
also there are no publications that compares the product and by-product
media.
coupling of supply and demand of amino acids in the cell, thus in a recombinant
reaction rate limitations in the bioreaction network for SAP production as the
controlling amino acids. In this case, supply of the controlling amino acids to the
7
In this context, in this study, first of all the effects of aspartic acid group amino
acids, which were reported to be the potential bottleneck in SAP synthesis (Çalık
for SAP production were determined in defined and complex media. Lastly, the
results were compared, in order to analyze the differences between defined and
complex media and to find out the potential strategies in order to increase SAP
and cell yields are much higher in complex medium, than that of defined
medium.
8
CHAPTER 2
LITERATURE SURVEY
2.1 Enzymes
and specificity. They are so effective as biological catalysts that most of the
reactions they catalyze would not proceed in a reasonable time without extremes
reactions. They lower the activation energy of the catalyzed reaction by binding
chemical catalysts. Foremost amongst these are their specificity and selectivity
not only for particular reactions but also in their discrimination between similar
pressure and pH. This decreases the energy requirements, reduces the capital
9
Enzymes, like other proteins, have molecular weights ranging from about 12,000
to over 1 million. Some enzymes require no chemical groups other than their
amino acid residues for activity. Others require an additional chemical group
called a cofactor. The cofactor may be either one or more inorganic ions, such as
Enzymes have been classified into six main types, depending on the nature of
the reaction catalysed. These groups are further subdivided according to the
developed by Enzyme Commission based on this division and the prefix E.C. is
generally employed with the numerical scheme (Blanch and Clark, 1997). For
instance the E.C. number of serine alkaline protease is EC 3.4.21.14. Table 2.1
10
2.2 Proteolytic Enzymes
Microbial proteases are classified into two major groups -peptidases and
proteinases- on the basis of their nature of attack (Moon and Parulekar, 1991).
and amino acids before cellular uptake. Proteases are classified by their catalytic
4. Metalloproteases (3.4.24)
The serine proteases are the most widely distributed group of proteolytic
enzymes of both microbial and animal origin. The enzymes have a reactive
serine residue in the active site and are generally inhibited by either diisopropyl
proteases have broad substrate specificities and are generally active at neutral
and alkaline pH, with an optimum between 7-11 (Moon and Parulekar, 1991).
They have generally low molecular weight in between 18.5-35 kDa. Most have
isoelectric points between pH 4.4 and 6.2. Serine proteases can be divided into
four sub-groups, according to their side chain specificity against oxidized insulin
11
These sub-groups are;
1. Trypsin-Like Proteases
2. Alkaline Proteases
4. Staphylococcal Proteases
Serine alkaline proteases (SAP) are one of the most important group of industrial
enzymes that are widely used in detergent, leather and meat industries. They
are produced by various bacteria, moulds and yeasts (Kalisz, 1988). They
account for approximately 35% of the microbial enzyme sales. The common
1. They all involve a particular serine residue that is essential for their catalytic
activity; and
The amino acid sequence of these enzymes depends on the microorganism that
they are produced by. However, whatever their amino acid composition is, they
fold in such a way that histidine, aspartic acid and serine form a catalytic triad.
Near the active site is a hydrophobic binding site, a slit-like pocket that
phenylalanine, tryptophane and leucine. SAP are sensitive to DFP and potato
inhibitor. They are most active at around pH 10 and their molecular weights are
in the 15-30 kDa range. The isoelectric point of SAP is normally around pH 9
12
2.3 Bioprocess Parameters for Serine Alkaline Protease Production
must be taken into account in order to have high product yield and selectivity.
These are; (1) microorganism, (2) medium composition and (3) bioreactor
2.3.1 Microorganism
obtain the desired product. The microorganism that is to be used should give
adequate yields, be able to secrete large amounts of protein and should not
suitable for industrial fermentations and produce large cell mass per volume
The rod shaped bacteria that aerobically form refractile endospores are assigned
to the genus Bacillus. The endospores of the bacilli are more resistant than the
vegetative cells to heat, drying, disinfectants, and other destructive agents and
thus may remain viable for centuries. Cell strain Gram positive and are motile by
Some species are strictly aerobic, others are facultatively anaerobic. Although
the majority are mesophobic, there are also psycrophilic and thermophilic
species. Some are acidophiles while others are alkalophiles. Strains of some
mineral salts, others need additional growth factors or amino acids, and still
Lechevalier, 1973). Bacilli are well known for their ability to excreate enzymes
13
such as amylases and proteases and are, therefore, excellent candidates for
1. It is non-pathogenic,
4. It can be grown more easily and has greater rates of protein synthesis than
many eucaryotic systems,
5. Its ability to secrete a wide variety of proteins far exceeds than of its
prokaryotic competitor, E.coli,
In the literature, Hanlon et al. (1981, 1982), Frankena et al. (1985, 1986), van
Putten et al. (1995, 1996) and Çalık et al. (1998, 2000) used B.licheniformis;
Hageman et al. (1984), Kole et al. (1988) used B.subtilis, Levisohn and Aronson
(1967, 1971) used B.cereus, Wright et al. (1992) used B.brevis and Moon and
Carbon and energy sources and their concentrations are indeed important as
they are tools for bioprocess medium design (Çalık et al., 2001). Culture
medium supplies the microorganism with all the essential elements for growth.
14
Certain microorganisms are capable of synthesizing all of their cellular
vitamins, etc.). The culture conditions that promote production of enzymes like
formulate a medium that is optimum for both cell growth and product formation.
Formation of macromolecules which constitute the major part of the cell mass
of the building blocks (Nielsen and Villadsen, 1994). Table 2.2 summarizes the
cells contain other metabolites in the form of inorganic salts (e.g., NH4+, PO43-,
K+, Ca2+, Na+, SO42-), metabolic intermediates (e.g., pyruvate, acetate), and
vitamins. A typical bacterial cell is composed of 50% carbon, 20% oxygen, 14%
K+, Ca2+, Na+, Mg2+, Cl- , and vitamins (Table 2.3) (Shuler and Kargi, 2002).
15
Table 2.2 The composition of a bacteria (E.coli)
Carbon 50
Oxygen 20
Nitrogen 14
Hydrogen 8
Phosphorus 3
Sulfur 1
Potassium 1
Sodium 1
Calcium 0.5
Magnesium 0.5
Chlorine 0.5
Iron 0.2
All others ≈ 0.3
16
Nutrients required by the cells can be classified into two categories (Shuler and
Kargi, 2002):
17
Alkaline protease is comprised of 53.8 % carbon and 15.6 % nitrogen.
Table 2.5 Some carbon and nitrogen sources utilized by fermentation industry
upon the special needs of particular bacteria a large variety and types of culture
media have been developed with different purposes and uses (Todar, 2000).
There are two major types of media depending on their composition or use.
18
A chemically defined (synthetic) medium is one in which the exact chemical
chemical constitution of the medium is not known. Defined media are usually
preferred since the attainable enzyme activity and cell yields are much higher
than that of defined media due to the presence of necessary growth factors,
vitamins, hormones, and trace elements. In this study, the components of the
complex medium are glucose and defatted soybean which was reported to have
the highest SAP activity than the other complex medium components (Özdemir,
2003).
Glucose
Soybean
three different grades according to the fat content: full-fat meal with a minimum
of 18 % fat, low fat meal with 4.5-9 % fat, and defeated meal with a maximum
of 2 % fat. The amino acid content and inorganic content of soybean are
19
Table 2.6 Amino acid content of soybean (Cejka, 1985)
Constituent Percentage
Potassium 1.67
Sodium 0.34
Calcium 0.28
Magnesium 0.22
Phosphorus 0.66
Sulfur 0.41
Chloride 0.024
Iodine 0.000054
Iron 0.0097
Copper 0.0012
Manganese 0.0028
Zinc 0.0022
Aluminum 0.0007
Carbon and energy sources and their concentrations are indeed important as
they are tools for bioprocess medium design (Çalık et al.,2001). Bacillus strains
can utilize alanine (Ala), arginine (Arg), asparagines (Asn), aspartate (Asp),
(Orn), proline (Pro), threaonine (Thr), and valine (Val) as the nitrogen source;
and Ala, Arg, Glu, Gln, His and Pro as the carbon source (Sonenshein, 1993).
Besides these amino acids, Bacillus strains can also use organic acids like citric,
acetic, succinic, pyruvic and α-ketoglutaric acids; glucose and the other hexoses;
20
In the literature there are a lot of studies on protease production by using
different liquid media. Hanlon and Hodges (1981) used a medium containing
(kg m-3) : 0.015, glucose; 0.04, Na2HPO4.12H2O; 0.026, KH2PO4; 0.01 NH4Cl;
Frakena et al. (1985) used (kg m-3): 1.8, glucose; 10.1, K2HPO4; 1.2, KH2PO4;
2.0, NH4Cl; 0.2, MgSO4.7H2O; 2.2x10-3, CaCl2; and citric acid, MnCl2, CoCl2,
limited chemostat cultures. Frakena et al. (1986) also investigated the effects of
citric acid as the carbon source at two concentrations that are 10 and 20 mM.
Moon and Parulekar (1991) used the medium reported by Frakena et al. (1985)
for their parametric study for B.firmus and investigated the effects of important
nitrogen and phosphorous sources and yeast extract on cell growth, synthesis
and secretion of protease. Write et al. (1992) used polypeptone, inorganic salts,
glucose or fructose for B.brevis and studied the enhancement and regulation of
Hübner et al. (1993) used (kg m-3): 12, glucose.H2O; 10, casein peptone; 5,
yeast extract; 1.6, (NH4)2HPO4; 0.5, Na2HPO4; 0.3, K2HPO4; 0.2, MnSO4.4H2O;
techniques for the production of alkaline protease for B.licheniformis. Çalık et al.
(1998) used a defined medium containing (kg m-3): 9, citric acid; 4.7,
21
the effects of initial citric acid concentration (CC) on growth and SAP activity and
acid concentration for maximum SAP activity was reported as CC= 9.0 kg m-3.
Although the activity obtained with citrate (Çalık et al., 2000) was higher than
that of glucose (Çalık et al., 2002) the enzyme was not able to maintain its high
activity; therefore, Çalık et al. (2002) used glucose as the carbon source.
Following this study, in order to increase SAP production Çalık et al. (2003a)
resulting SAP activity. Among the recombinant species the highest SAP
the same study they also investigated the effects of oxygen transfer in SAP
production process in larger scale together with the oxygen transfer parameters.
In the literature there are no studies related with the influence of aspartic acid
in SAP synthesis, and also there are no publications that compares the product
complex media.
22
2.3.3 Bioreactor Operation Parameters
constant and at their optimal values throughout the fermentation process. The
the growth process is the result of many enzymatic processes the influence of
both culture parameters on the overall bioreaction is quite complex (Çalık et al.,
2001).
denaturation starts, and a rapid decrease beyond this temperature (Nielsen and
Villadsen, 1994).
species across the cell membrane. Variation in pH alters acid-base equilibria and
fluxes of various nutrients, inducers and growth factors between the abiotic and
biotic phase (Moon and Parulekar, 1991). The influence of pH on cellular activity
the total enzyme activity of the cell is therefore a complex function of the
23
environmental pH. Microbial cells have a remarkable ability to maintain the
value is 7.5 (Çalık et al., 2001). Frankena et al. (1986), Kole et al. (1988),
Moon and Parulekar (1991), and Wright et al. (1992) studied protease
(1996) reported the results obtained with uncontrolled-pH operation without any
explanation or discussion. Çalık et al. (1998, 2000) reported the time course
conditions in a wide range. Çalık et al. (2002a) used glucose as the sole carbon
on the basis of the findings of the previous study, the same research group
reaction network rates whereupon the rate limiting step of the bioprocess. In all
the reported works in the literature, the temperature is between 35-40 °C.
Hübner et al., (1993) and van Putten et al.(1996), at initial pH=6.8, T=39.5°C
and Çalık et al.(1998, 2000) at pH=7.25, T=37°C. Among these studies only
Moon and Parulekar (1991) investigated the controlled pH effect and reported
optimum pH as pH=7.7.
24
2.3.3.2 Oxygen Transfer
analysis some bioprocesses require high oxygen transfer rate conditions while
others require controlled oxygen transfer rate conditions (Çalık et al., 1999). It
has been extensively investigated in defined (Çalık et al. 1998, 1999, 2000) and
molasses based complex medium (Çalık et al. 2003) for serine alkaline protease
Cellular growth and product formation are the result of a very large number of
chemical reactions that occur inside individual cells and involves transport of
substrates into the cell, followed by conversion of the intracellular substrates into
biomass and metabolic products and then the metabolic products are excreted
back into the extracellular medium. Cellular processes can therefore be divided
25
p
abiotic phase
s S P+X
biotic phase
central metabolic pathways are few in number and are remarkably similar in all
two stages: a hexose stage, in which ATP is consumed, and a triose phase, a net
also known as hexose monophosphate shunt. This pathway has both aerobic and
anaerobic parts. Pentose phosphate pathway provides nucleotide, RNA and DNA
26
Tricaboxylic acid cycle, whose driving force is oxygen, is the major pathway of
which pyruvate is completely oxidized to CO2. TCA cycle functions not only as a
process for energy generation but also a supply of precursors for biosynthesis of
amino acids and nucleotides. In TCA cycle there are two key intermediates that
are used in the synthesis of necessary amino acids for SAP production (Horton,
1992). From OA; aspartic acid group amino acids, from α-ketoglutarate glutamic
coupling of supply and demand of amino acids in the cell, thus in a recombinant
reaction rate limitations in the bioreaction network for SAP production as the
controlling amino acids. In this case, supply of the controlling amino acids to the
The aspartic acid group amino acids constitute 26 % of the total amino acids in
serine alkaline protease and they play a critical role not only in growth and
protease synthesis (Çalık et al., 1999). Figure 2.2 presents the aspartic acid
pathway via which aspartic acid group amino acids are synthesized. Aspartic
acid group amino acids are produced via oxalo acetic acid. After aspartate is
27
produced the pathway splits into two branches, from one branch asparagine and
from the other branch aspartate semialdehyde are produced. After aspartate
semialdehyde the pathway again splits into two brances. From one branch
lysine; from the other branch methionine, threonine and isoleucine are
synthesized.
L-Aspartate
L-Asparagine
Aspartate semialdehyde
Homoserine L-Methionine
L,2,3 Dihydrodipicolinate
L-Threonine L-Isoleucine
L,2,3,4,5 Tetrahydrodipicolinate
meso-Diaminopimelate
L-Lysine
28
CHAPTER 3
3.1 Microorganism
T=-20°C.
agar slants under sterile conditions and they were incubated at 30°C for 36h.
Table 3.1 gives the composition of solid medium for serine alkaline protease
agitation rate of N=200 rpm for 5.5 to 6 hours (until an absorbance of 0.28-0.35
29
at 600 nm was reached). Microorganism growth was conducted in orbital shakers
under agitation and heating rate control, using 150 ml air-filtered Erlenmeyer
medium for cell growth and enzyme production is given in Table 3.2 (Çalık,
1998).
Table 3.1 The composition of solid medium for recombinant Bacillus sp.
MnSO4.2H2O 0.01
Agar 15.0
Peptone 2.5
Azocasein 2.0
Chloramphenicol 0.007
Table 3.2 The composition of precultivation medium for recombinant Bacillus sp.
MnSO4.2H2O 0.01
Peptone 5.0
Soytryptone 15.0
Na2HPO4 0.25
CaCl2 0.1
Chloramphenicol 0.007
30
3.4 Production Medium
conducted in orbital shakers under agitation (N=200 rpm) and heating rate
(37°C) control, using 250 ml air-filtered Erlenmeyer flasks with a working volume
Bacillus sp.
Glucose 8.0
(NH4)2HPO4 4.71
KH2PO4 2.0
NaH2PO4 5.63
Na2HPO4.2H2O 0.055
Mg(CH3COO)2 0.87
CaCl2 0.2
Chloramphenicol 0.007
31
Table 3.4 The composition of reference complex medium for recombinant
Bacillus sp.
Soybean 20
Glucose 8.0
NaH2PO4 2.815
Na2HPO4.2H2O 0.028
Chloramphenicol 0.007
3.5 Analysis
During the bioprocess, samples were taken at characteristic cultivation times and
cell concentration, SAP activity, glucose, amino acid, organic acid concentrations
and also DNA and RNA concentrations were determined. Figure 3.1 summarizes
Sample
Analysis Analysis
- SAP activity - DNA comcentration
- Glucose concentration - RNA concentration
- Extracellular amino - Intracellular amino
acid concentrations acid concentrations
- Extracellular organic - Intracellular organic
acid concentrations acid concentrations
32
3.5.1 Cell Concentration
(Çalık, 1998).
Proteolytic activity was measured by hydrolysis of casein. The culture broth was
0.5% w/v) in borate buffer was mixed with 1 ml of diluted bacterial broth and
hydrolyzed under T= 37°C, pH=10 for 20 min. The reaction was stopped by
adding 10 % (w/v) trichloroacetic acid (TCA) and the reaction mixture was
calibration curve (Appendix B). One unit protease activity was defined as the
DNS method (Miller, 1959) tests for the presence of free carbonyl group (C=O),
the so-called reducing sugars. This involves the oxidation of the aldehyde
(Appendix C). For every newly prepared DNS solution, calibration curve should
33
1. DNS solution was added to 1 cm3 of diluted sample.
2. This mixture was placed into a boiling water bath and heated for 5 min.
3. After 5 min the mixture was placed into an ice bath and cooled for 5 min.
through the same steps but do not contain any reduced sugar.
Amino acid concentrations were measured with an amino acid analysis system
(Waters, HPLC), using the Pico Tag method (Cohen, 1983). The method is based
program developed for amino acids. The amino acid concentrations were
amino acids solution. The analysis was performed under the conditions specified
below:
Column temperature : 38 °C
Injection volume : 4 µl
34
3.5.5 Determination of Organic Acids Concentrations
analyzed at 20kV and 15°C with a negative power supply by hydrostatic pressure
0.5mM OFM Anion Bt (Waters) as the flow modifier at pH=5.6 (for α-ketoglutaric
acid, acetic acid, malic acid, fumaric acid, succinic acid, lactic acid, oxalacetate
and gluconic acid) and at pH=7.0 (for, pyruvic acid, citric acid, lactic acid,
amino acid and organic acid concentrations.). After a washing step with distilled
water (4 °C) and 0.009 M NaOH solution (4 °C) the biomass was hydrolyzed
using 750 µL, 6 M HCl at 105°C for 18 h. After the hydrolysis, 525 µL isopropanol
was added and the sample was kept at -20°C for 20 min in order to accelerate
the precipitate of DNA and RNA. Thereafter, the sample was centrifuged at
12000 g and at T=0°C. The supernatant was stored at -20°C for the
precipitate was used to determine DNA and RNA concentrations; for this aim
35
3.5.6.1 Determination of DNA Concentration by Diphenylamine Reaction
The procedure involves chemical hydrolysis of DNA: when heated (e.g. 95°C) in
bond linking the base to the sugar is hydrolyzed with subsequent hydrolysis of
curve, which gives the exact relationship between A600 and DNA concentration.
A. Supplies
* 1.6 M HClO4
acetic acid+2 ml conc. sulfuric acid (use fume hood to make stock solution)
* TE buffer (Appendix E)
B. Procedure
A set of reaction tubes was marked (tape and Sharpie should be used; marking
were placed into the tubes and diluted to 1 ml with distilled water. Then 1 ml
acid was added to each and heated for 15 min at 95oC; thereafter the tubes
spectrophotometer was turned on and is zeroed in at 540 nm; water was used
as a blank. After 5 min diphenylamine reagent was added and the tubes were
36
transferred to a boiling water boiling water bath for 10 min. The tubes were then
cooled in an ice bath (≈5 min). The absorbance was read at 540 nm for each
A. Supplies
B. Procedure
1. After the biomass was hydrolyzed and DNA and RNA were precipitated by the
in TE buffer.
2. 100 µL of the sample was mixed with 200 µL distilled water then 100 µL
3. This mixture was heated in a boiling water bath for 20 min then cooled in an
through the same steps and contain 100 µL distilled water instead of the sample.
5. 500 µL of this reaction mixture was added to 2000 µL n-butanol and the
37
absorbance was measured at 670 nm. The absorbance was related to the RNA
relationship between A670 and RNA concentration. The calibration curve for RNA
is given in Appendix G.
Concentrations
After precipitating the DNA and RNA, the supernatent was dried at 60 °C then
dissolved in 100 µL acetonitryl and by using the methods given in part 3.5.4 and
38
CHAPTER 4
Serine alkaline protease (SAP) synthesis depends on good coupling of supply and
demand of amino acids in the cell, the synthesis of which are dependent on the
reaction rate limitations in the bioreaction network for SAP production as the
controlling amino acids. In this case, supply of the controlling amino acids to the
fermentation broth and supplied period are indeed important. In this study, at
first the effects of supply of aspartic acid group amino acids, which were
thereafter, the product and by-product distributions of the bioprocess for SAP
production were determined in defined and complex media. Lastly, the results
complex media and to find out the potential strategies in order to increase SAP
39
and cell yields are much higher in complex medium, than that of the defined
medium.
The effects of initial aspartic acid concentration on growth and SAP activity were
0-15mM by using the reference defined production medium given in Table 3.3.
Variation in biomass concentration and SAP activity is given in Figure 4.1. The
cell growth and the cell concentrations obtained at the end of the fermentation
were not affected with the supply of aspartic acid indicating that aspartic acid is
1074 U cm-3 which was 1.87-fold higher than that obtained for the reference
medium indicating that in SAP synthesising period the amount of the aspartic
acid is important as it is the precursor of the aspartic acid group amino acids;
and aspartic group amino acid content is 26 % of the total amino acids in the
SAP molecule. With the further increase in aspartic acid concentration SAP
4.1).
The effect of initial asparagine concentration on growth and SAP activity were
15mM by using the reference defined production medium given in Table 3.3.
40
asparagine didn’t change the biomass concentration considerably,higher
(0.25, 0.5, 1.0 mM), SAP activity decreased with the increase in asparagine
concentration. The highest SAP activity was obtained as 1082 U cm-3 for an
order to keep the asparagine concentration at a certain level. For this purpose,
activity is 1.89-fold higher than the reference medium activity. On the other
aspartic acid didn’t affect cell growth. In contrast to the aspartic acid results,
in SAP production.
2,50 1800
1600
2,00 1400
1200
CX , g/dm3
A, U/cm3
1,50
1000
800
1,00
600
0,50 400
200
0,00 0
0 5 10 15 20 25 30 35 40 45
t, h
41
2,5 1800
1600
2,0
1400
1200
1,5
CX , g/dm 3
A, U/cm3
1000
800
1,0
600
400
0,5
200
0,0 0
0 10 20 30 40 50
t, h
The effects of initial isoleucine concentration on growth and SAP activity were
Figure 4.3. Cell growth was inhibited with the addition of isoleucine at t = 0 h,
fermentation medium at low concentrations (0.25, 0.5 and 1.0 mM) increased
SAP production, SAP activity was almost the same and greater than that of the
reference medium. The highest SAP activity was obtained as 813 U cm-3 for
medium activity. At isoleucine concentrations higher than 2.5 mM, SAP activity
decreased significantly below that of the reference medium probably due to the
42
2.50 1800
1600
2.00
1400
1200
1.50
A, U/cm3
CX , g/dm 3
1000
800
1.00
600
400
0.50
200
0.00 0
0 5 10 15 20 25 30 35 40 45
t, h
The effects of initial lysine concentration on growth and SAP activity were
Figure 4.4. Cell growth was slightly inhibited with the addition of lysine. At lower
lysine concentrations (0.25, 0.5 and 1.0 mM) SAP activity increased when initial
lysine concentration was decreased and a maximum SAP activity of 926 U cm-3
than the reference medium activity. On the other hand at CLyso≥2.5 mM SAP
activity was lower than that obtained for the reference medium and decreased
with the increase in the initial lysine concentration. Similar to asparagine results,
this indicates that with the addition of lysine alone to the medium at t=0 h, the
43
cells regulate themselves in order to keep the lysine concentration at a certain
level. For this purpose, the cell enzyme levels should be regulated in such a way
2.5 1800
1600
2
1400
1200
1.5
A, U/cm 3
CX , g/dm 3
1000
800
1
600
400
0.5
200
0 0
0 5 10 15 20 25 30 35 40 45
t,h
The effects of initial threonine concentration on growth and SAP activity were
concentration and SAP activity are given in Figure 4.5. Cell growth and cell
concentrations at the end of the fermentation were higher than that in the
0.25 and 0.5 mM had similar trends in SAP activity profiles. Moreover, at the
44
concentration range of 1.0-15mM SAP activities were almost the same with the
reference medium. The highest SAP activity was obtained as 900 U cm-3 with
an initial threonine concentration of 0.25 mM. This activity was 1.48-fold higher
2.5 1800
1600
2
1400
1200
A, U/cm3
1.5
CX , g/dm3
1000
800
1
600
400
0.5
200
0 0
0 10 20 30 40 50
t,h
The effects of initial methionine concentration on growth and SAP activity were
concentration and SAP activity are given in Figure 4.6. Cell concentrations were
higher than that in the reference medium before t = 23 h but then started to
45
SAP activity was greater than that obtained for the reference medium and the
mM. This activity was 1.4-fold higher than that obtained in the reference
3 1800
1600
2.5
1400
2 1200
A, U/cm3
1000
CX, g/dm3
1.5
800
1 600
400
0.5
200
0 0
0 10 20 30 40 50
t,h
The product and by-product distributions of the bioprocess for SAP production
were analyzed in both defined and complex media in order to compare, and
46
4.7.1 Variation in Glucose and Cell Concentrations in Defined Medium
The variations in glucose -that enters into the carbon metabolism from the
cultivation time are presented in Figure 4.7. It can be seen from the figure that
glucose was consumed at a high rate in the first 5 h of the bioprocess, then up
to 40 h the consumption rate was almost the same and after 40 h the
high rate between t= 0-5 h, and reached to its maximum value 1.75 g/dm3
presented in Figure 4.8. Both specific glucose consumption and growth rates
decrease with the cultivation time. After t=10 h specific glucose consumption
then after t=30 h as the cell concentration did not change the specific growth
9 2
8 1.8
7 1.6
6
1.4
CX , g/dm 3
CG , g/dm 3
1.2
5
1
4
0.8
3
0.6
2
0.4
1 0.2
0 0
0 10 20 30 40 50 60
t,h
Figure 4.7 Variations of glucose (¡) and cell () concentrations in defined
medium with the cultivation time
47
2.5 0.35
0.3
2
0.15
1
0.1
0.5
0.05
0 0
0 10 20 30 40 50 60
t, h
Figure 4.8 Variations in specific glucose consumption (¡) and specific growth
() rates in defined medium with the cultivation time
The variations of serine alkaline protease activity (A) and specific serine alkaline
activity (A') and specific serine alkaline protease production rate (rp) with the
cultivation time are shown in Figure 4.9 and 4.10, respectively. During the cell
formation period (t= 0-8 h), the rate of increase in serine alkaline protease
activity was low. After t= 8 h serine alkaline protease activity started to increase
corresponds to the stationary phase of the growth since serine alkaline protease
maximums at t=50 h. The highest serine alkaline protease activity was obtained
48
800 600000
700
500000
600
400000
500
A', U / gX
3
A , U/cm
400 300000
300
200000
200
100000
100
0 0
0 10 20 30 40 50
t,h
Figure 4.9 Variations in SAP activity ( ) and specific SAP activity ({) in defined
medium with cultivation time
25000
20000
15000
-1
rP , U g X h
10000
5000
0
0 10 20 30 40 50
t,h
Figure 4.10 Variation in specific SAP production rate in defined medium with
cultivation time
49
4.7.3 Variations in the Extracellular Amino Acid and Organic Acid
phenyalanine, proline and tyrosine in the fermentation broth. Since there were
no amino acids in the reference defined medium, these amino acids should be
excreted to the broth during the growth in precultivation medium and supplied
the fermentation broth while cysteine and tyrosine initially present were utilized.
concentration decreased with the cultivation time therefore, one can conclude
that ornithine was supplied by the cells from the fermentation broth and utilized
the range between 0.082-0.134 g dm-3 and 0.3-0.6 g dm-3, respectively. Within
the time interval between t=10-20h and at t=50 h cysteine was detected in the
amino acids was present in the extracellular medium throughout the whole
bioprocess. This indicates that proline was being sufficiently synthesized by the
cell that the excess of this amino acid was excreted to the medium and since the
total glutamic acid content is only 8 % of the total amino acids in SAP molecule,
glutamic acid group amino acids may not be a potential bottleneck in SAP
production.
Table 4.2 shows the variations in the extracellular organic acid concentrations.
As it is seen from the table the only organic acid excreted to the fermentation
broth was oxalo acetic acid – the branch point for aspartic acid group amino
50
Oxaloacetic acid is synthesized either from the glycolyisis pathway metabolites,
TCA cycle metabolite malic acid; and used for the energy generation in the TCA
cycle and in the synthesis of aspartic acid. The existence of oxalo acetic acid in
the fermentation broth denotes the insufficient operation of the TCA cycle
cultivation time, h
g/L 0 5 10 15 20 25 30 35 40 45 50
Ala 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Asp 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Asn 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Arg 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cys 0.023 0.0 0.054 0.012 0.018 0.0 0.0 0.0 0.0 0.0 0.028
Glu 0.0 0.055 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Gly 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
His 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Ile 0.0 0.0 0.0 0.0 0.0 0.005 0.0 0.0 0.0 0.0 0.0
Leu 0.0 0.0 0.0 0.0 0.0 0.005 0.0 0.0 0.0 0.0 0.0
Met 0.032 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Orn 0.24 0.213 0.15 0.176 0.14 0.092 0.088 0.058 0.090 0.06 0.0
Phe 0.134 0.088 0.087 0.087 0.085 0.084 0.125 0.106 0.130 0.126 0.082
Pro 0.320 0.344 0.313 0.396 0.490 0.4 0.4 0.39 0.52 0.560 0.662
Ser 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Thr 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Trp 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tyr 0.005 0.0 0.0 0.0 0.0 0.0 0.0 0.002 0.0 0.0 0.0
Val 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
51
Table 4.2 Variation in the extracellular organic acid concentrations in defined
medium
cultivation time, h
g/L*106 5 15 20 25 30 35 40
OA 261 253 230 0 0 0 207
Suc 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Pyr 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Mal 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Lac 0.0 0.0 0.0 0.0 0.0 0.0 0.0
KG 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Gluc 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Glu 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cit 0.0 0.0 0.0 0.0 0.0 0.0 0.0
But 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Asp 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Ac 0.0 0.0 0.0 0.0 0.0 0.0 0.0
In the bioprocess for serine alkaline protease production, the intracellular amino
the amino acid(s) creates reaction rate limitations in the bioreaction network as
the controlling amino acids. Table 4.3 shows the variations of intracellular amino
acid concentrations with the cultivation time in defined medium. At t=0 h there
were isoleucine, leucine, methionine and phenylalanine in the cell. In the first 5h
of the process; intracellular isoleucine and leucine were consumed; on the other
proline and tyrosine were present in the cell at low concentrations; but
Moreover, phenyalanine was detected in high amounts in the cell throughout the
medium at t=0 h. At the onset of SAP synthesis (t=10 h) all of the amino acids
52
were present in the cell except histidine, isoleucine, leucine, tryptophan and
appeared at higher concentration in the cell than the others at t=10 h. Between
t=15 h and t=35 h the amino acids concentrations were low, due to their
phenylalanine and proline were present both in the extracellular and intracellular
media throughout the whole bioprocess. Serine and valine, which are 11.6 and
11.3 % of the amino acid content of SAP, were neither observed in the cell nor in
the extracellular medium during the bioprocess. Isoleucine and leucine were
present at t=0 h and t=40 h in the intracellular medium, and only at t=25 h in
the fermentation broth indicating that these amino acids were synthesized and
then consumed by the cell for growth and product formation while with the
decrease in cell functions the consumption rate of these amino acids should be
lower than the production rate; consequently produced more than the demand of
the cells. Alanine (14.5 % of the total amino acid content of SAP), was observed
in low amounts in the intracellular medium only at t=5, 10 and 40 h; while, was
not detected in the extracellular medium. At t=40 h, where the serine alkaline
protease activity was about to reach its maximum value, all of the amino acids
were present in the cell except alanine, cysteine, glysine, histidine, serine,
tryptophan and valine. As with the decrease in cell functions still these amino
acids were not observed in the fermentation broth, the synthesis of these amino
In this study variations in the intracellular organic acid concentrations with the
cultivation time were also determined. As it is seen from Table 4.4 lactic acid
was present in the cell throughout the whole bioprocess and oxalic acid was
detected in the cell in the exponential phase of SAP production and t=40 h. The
53
existence of these organic acids in the cell indicates the insufficient operation of
cultivation time, h
6
g/g*10 0 5 10 15 20 25 30 35 40 45 50
Ala 0.0 0.11 1.66 0.0 0.0 0.0 0.0 0.0 0.0 0.23 0.0
Asp 0.0 0.0 7.63 0.0 0.0 0.0 0.0 0.0 0.49 0.0 0.0
Asn 0.0 0.0 7.57 0.0 0.0 0.0 0.0 0.0 0.49 0.0 0.0
Arg 0.0 0.0 1.77 0.0 0.0 0.0 0.0 0.0 0.13 0.0 0.0
Cys 0.0 0.0 5.32 0.0 0.0 0.0 0.0 0.0 0.00 3.23 0.0
Glu 0.0 0.0 30.66 0.0 0.0 0.0 0.0 0.0 1.40 0.67 0.0
Gly 0.0 0.0 4.14 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
His 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Ile 1.46 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.94 0.0 0.0
Leu 1.46 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.94 0.0 0.0
Met 1.08 0.99 21.50 91.13 74.02 0.77 0.0 0.0 4.32 3.92 2.02
Orn 0.0 0.0 31.56 16.73 34.32 33.39 18.57 0.0 35.56 37.49 35.83
Phe 10.46 11.84 13.88 11.36 11.36 11.42 10.80 9.56 12.60 12.97 15.08
Pro 0.0 3.66 6.41 4.16 4.84 4.90 4.93 4.54 4.57 4.79 4.36
Ser 0.0 0.0 1.41 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Thr 0.0 0.0 1.21 0.0 0.0 0.0 0.0 0.0 0.09 0.0 0.0
Trp 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tyr 0.0 0.29 8.01 0.25 0.0 0.0 0.0 0.0 0.73 0.62 0.25
Val 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
cultivation time, h
g/g*10 6 5 15 20 25 30 35 40
OA 0.0 0.012 0.011 0.0 0.0 0.0 0.009
Suc 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Pyr 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Mal 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Lac 0.0 0.007 0.005 0.003 0.004 0.003 0.001
KG 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Gluc 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Gluc 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cit 0.0 0.0 0.0 0.0 0.0 0.0 0.0
But 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Asp 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Ac 0.0 0.0 0.0 0.0 0.0 0.0 0.0
54
4.7.5 Variations in Glucose and Cell Concentrations in Complex Medium
The variations in glucose and cell concentrations in complex medium with the
cultivation time are presented in Figure 4.11. Besides glucose soybean was also
As it is seen from the figure, glucose was consumed at a high rate in the first 5 h
of the bioprocess, then at t=5-30 h the consumption rate was almost constant
and after 30 h the glucose concentration remained constant till the end of the
constant. The highest cell concentration was reached at t=35 h and its value was
14.3 g dm-3. Variations of specific glucose consumption and growth rates are
presented in Figure 4.12. Both specific glucose consumption and growth rates
9 16
8 14
7
12
6
10
CX , g/dm3
5
CG , g/dm 3
8
4
6
3
4
2
1 2
0 0
0 10 20 30 40 50
t,h
Figure 4.11 Variations in glucose (¡) and cell () concentrations in complex
medium with the cultivation time
55
1.6 0.4
1.4 0.35
0.8 0.2
0.6 0.15
0.4 0.1
0.2 0.05
0 0
0 10 20 30 40 50 60
t, h
Figure 4.12 Variation in specific glucose consumption (¡) and specific growth
() rates in complex medium with the cultivation time
The variations of serine alkaline protease activity (A) and specific serine alkaline
protease activity (A') and specific serine alkaline protease production rate (rP)
with the cultivation time in complex medium are given in Figure 4.13 and 4.14,
and the highest serine alkaline protease activity was observed at t=50 h having
a value of 2015 U cm-3. Same trend was observed in the variation of specific SAP
production. Specific SAP production rate increased after t=10 h and a maximum
was observed at t=20 h, where glucose reached its limiting value indicating that
56
800 600000
700
500000
600
400000
500
3
A' , U / gX
A , U/cm
400 300000
300
200000
200
100000
100
0 0
0 10 20 30 40 50
t,h
Figure 4.13 Variations in SAP activity ( ) and specific SAP activity ({) in
complex medium with cultivation time
30000
25000
20000
rP , U g X-1 h-1
15000
10000
5000
0
0 10 20 30 40 50
t,h
57
4.7.7 Variations in the Extracellular Amino Acid and Organic Acid
In this study, soybean was used as a carbon source for complex medium which
extracellular amino acid concentrations are given in Table 4.5. In SAP production
with complex medium as the precultivation medium used for defined medium
complex medium at t= 0 h, all the amino acids were present in the extracellular
medium except alanine, glycine, and serine, that are not present in soybean
structure, and tryptophan, and valine. At t=5 h of the bioprocess, all the amino
acids present in the broth at the beginning of the fermentation were consumed
amino acids were not present in the fermentation broth at t= 5 h one can
conclude that the consumption rate of the soybean protein amino acids should
be either equal or higher than their liberation rates. At the beginning of SAP
phenylalanine and tyrosine were present in the fermentation broth, and the
consumption rate of the soybean protein amino acids should be either equal or
higher than their liberation rates. Glutamate and glutamine serve as the donor of
virtually all amino and amide groups in cellular components, either by the direct
58
biosynthesis of serine, alanine, valine, leucine, aspartate, meso-
Consequently glutamate, produced by the below reactions, was more than the
59
At t=20-30 glutamate, cysteine, ornthine, tyrosine and phenylalanine were
high and phenylalanine was further supplied from the soybean, and as tyrosine
that was present in the fermentation both although it was not present in the
than the demand of the cells since it was excreted to the fermentation broth.
fermentation and was between 0.11-0.33 g dm-3 At the time where SAP activity
glycine (12.7% of SAP), serine (11.6% of SAP), valine (11.3% of SAP), and
alanine (14.5 % of SAP) (except t=10 h and t= 25 h) were not observed in the
Table 4.6 shows the variations in the extracellular organic acid concentrations in
oxalic acid, succinic acid and lactic acid were excreted to the fermentation broth.
The presence of these organic acids indicates the oxygen limitation. Compared to
Table 4.7 presents the variations in the intracellular amino acid concentrations in
complex medium. As seen in the Table 4.7, at t=0 h there were isoleucine,
leucine, methionine and phenylalanine in the cell similar to the defined medium
since the same precultivation medium was used for complex medium
experiments. At t=5 h all of the amino acids were present in the intracellular
60
medium except cysteine, ornithine, serine, threonine, tryptophan and valine.
decreased with the cultivation time until the end of the exponential SAP
medium. Moreover methionine was not observed in the fermentation broth after
the beginning of the bioprocess. This indicates that methionine that was
liberated from soybean was supplied from the fermentation broth and not
synthesized by the cells. Alanine, isoleucine and leucine were also present in the
was present both in the cell and in the extracellular medium; whereas glycine
(except t=10 h), serine, tryptophan and valine were neither observed in the
intracellular medium nor in the fermentation broth. This denotes that, glycine,
serine, tryptophan and valine barely supply the requirement of the cell for
growth and product formation. At the time where serine alkaline protease
Table 4.8 presents the variations in the intracellular organic acid concentrations
oxalic acid and butyric acid were observed in the cell. During the bioprocess
oxalic acid was also present in the fermentation broth whereas, butyric acid was
61
Table 4.5 Variations in the extracellular amino acid concentrations in complex
medium
cultivation time, h
g/L 0 5 10 15 20 25 30 35 40 45 50
Ala 0.0 0.0 0.0004 0.0 0.0 0.0007 0.0 0.0 0.0 0.0 0.0
Asp 0.004 0.0 0.0 0.0 0.0036 0.0 0.0005 0.009 0.0 0.0012 0.0
Asn 0.004 0.0 0.0 0.0 0.0035 0.0 0.0004 0.009 0.0 0.0012 0.0
Arg 0.01 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cys 0.02 0.009 0.0 0.0 0.045 0.039 0.0474 0.0 0.0975 0.017 0.046
Glu 0.01 0.008 0.058 0.017 0.047 0.007 0.006 0.003 0.0067 0.0016 0.0
Gly 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
His 0.0 0.0 0.0 0.0 0.0170 0.0 0.0 0.0008 0.0 0.0 0.0
Ile 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Leu 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Met 0.02 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Orn 0.33 0.12 0.113 0.12 0.26 0.112 0.12 0.0 0.15 0.27 0.13
Phe 0.11 0.19 0.098 0.13 0.12 0.122 0.14 0.0 0.16 0.27 0.14
Pro 0.0 0.039 0.0 0.0 0.0 0.0 0.0 0.04 0.0 0.0 0.0
Ser 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Thr 0.0068 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Trp 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tyr 0.0053 0.0 0.0063 0.0 0.0021 0.0023 0.0017 0.0 0.0023 0.0041 0.0
Val 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
cultivation time, h
6
g/L*10 5 15 20 25 30 35 40
OA 245 213 409 32 66.2 33.2 352
Suc 42 26 0.0 0.0 52.3 25.3 28.4
Pyr 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Mal 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Lac 0.0 0.0 20.5 196 0.0 0.0 0.0
KG 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Gluc 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Glu 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cit 0.0 0.0 0.0 0.0 0.0 0.0 0.0
But 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Asp 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Ac 0.0 0.0 0.0 0.0 0.0 0.0 0.0
62
Table 4.7 Variations in the intracellular amino acid concentrations in complex
medium
cultivation time, h
6
g/g*10 0 5 10 15 20 25 30 35 40 45 50
Ala 0.0 1.42 0.11 0.18 0.04 0.0 0.0 0.0 0.0 0.0 0.0
Asp 0.0 0.55 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Asn 0.0 0.55 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Arg 0.0 0.48 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.19
Cys 0.0 0.00 3.93 0.0 0.0 0.44 0.0 0.0 0.0 0.0 0.0
Glu 0.0 1.53 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Gly 0.0 0.27 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
His 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Ile 1.46 4.75 0.0 3.38 0.41 0.0 0.0 0.0 0.0 0.0 0.0
Leu 1.46 4.75 0.0 3.38 0.41 0.0 0.0 0.0 0.0 0.0 0.0
Met 1.08 5.34 2.89 1.88 0.37 0.17 0.0 0.0 0.0 0.0 0.51
Orn 0.0 0.0 47.28 48.88 44.98 42.45 35.49 39.51 16.23 40.37 29.02
Phe 10.46 10.61 7.15 10.05 0.0 0.0 0.0 0.0 9.57 0.0 13.68
Pro 0.0 1.83 0.14 0.00 0.05 0.0 0.0 0.0 3.90 0.0 4.24
Ser 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00
Thr 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.13
Trp 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tyr 0.0 2.51 1.56 0.75 0.18 0.19 0.0 0.24 0.0 0.27 0.0
Val 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
cultivation time, h
6
G/g*10 5 15 20 25 30 35 40
OA 0.093 0.009 0.014 0.002 0.003 0.009 0.054
Suc 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Pyr 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Mal 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Lac 0.0 0.0 0.0 0.0 0.0 0.0 0.0
KG 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Gluc 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Gluc 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cit 0.0 0.0 0.0 0.0 0.0 0.0 0.0
But 0.0 0.0016 0.0023 0.0 0.0 0.0 0.0
Asp 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Ac 0.0 0.0 0.0 0.0 0.0 0.0 0.0
63
4.7.9 Variations in the DNA and RNA Concentrations in Defined and
Complex Media
Variations of DNA and RNA concentrations with the cultivation time were
concentrations vary between 0.07-0.20 gDNA gX-1 and RNA concentrations were in
the range of 0.1-0.29 gRNA gX-1. In complex medium DNA concentrations were
between 0.07-0.20 gDNA gX-1 and RNA concentrations vary between 0.06-0.21
gRNA gX-1.
The product and by-product distributions of the bioprocess for SAP production
were analyzed in both defined and complex media in order to compare and
In complex medium glucose consumption rate was higher than that of defined
medium and glucose reached to the limiting value earlier than defined medium.
The specific glucose consumption rate decreased with the cultivation time both in
defined and complex media. However, the decrease in the glucose consumption
rate was faster in complex medium than the defined medium. Maximum specific
glucose consumption rates in defined medium and complex medium were 1.97
and 1.42 gG gX-1 h-1, respectively. The maximum specific glucose consumption
rate was lower in complex medium since there was an alternative carbon source,
t=5 h and thereafter remained constant. On the other hand in complex medium,
64
14.3 g dm-3 in complex medium. The reason for such an increase in cell
carbon source. Both in defined and complex media the specific growth rate
decreased with the cultivation time. The maximum specific growth rates in
defined medium and complex medium were 0.3 and 0.36 h-1, respectively. The
increase in the specific SAP production rate was also higher in complex medium
than that in defined medium. The highest SAP activity obtained in complex
medium was 3–fold higher than the SAP activity in defined medium as expected
should secrete protease. In both defined and complex media, glycine (12.7 % of
the total amino acids in SAP), serine (11.6 % of the total amino acids in SAP)
and valine (11.3 % of the total amino acids in SAP) were neither observed in the
extracellular nor in the intracellular media indicating that these amino acids were
also conclude that the synthesis of these amino acids can be a rate-limiting step.
(Çalık et al., 1999) was not present in the fermentation broth and was observed
only at t=10 h and t=40 h in the cell in defined medium. On the other hand, in
and between t=20-45 h in the fermentation broth and t=5 h in the cell.
Methionine -that initiates protein synthesis- was observed both in defined and
of the bioprocess. This indicates that methionine was supplied from the
fermentation broth and did not synthesized by the cells. On the other hand in
65
t=15 h, gave a maximum and decreased thereafter. Furthermore, methionine
bioprocess indicating that methionine was synthesized by the cells and did not
supplied from the fermentation broth in defined medium. Both methionine and
asparagine are in the aspartic acid group amino acids. In the aspartic acid
pathway (Figure 2.2) from one branch asparagine and from another branch
defined medium asparagine was neither present in the cell (except t=10 h and
t=40 h) nor in the fermentation broth however, methionine was observed in the
cell in high amounts in defined medium. This may indicate that the flux towards
asparagine might be low compared to the flux values of the other branches in
medium. In both defined and complex media, oxaloacetic acid was observed
acid in the extracellular medium indicating that the operation of TCA cycle was
insufficient. In defined medium lactic acid was present in the cell throughout the
whole bioprocess whereas in complex medium lactic acid was observed only at
t=20-25 h in the fermentation broth. Excretion of lactic acid to the broth also
66
CHAPTER 5
Serine alkaline protease (SAP) synthesis depends on good coupling of supply and
demand of amino acids in the cell, thus in a recombinant bacilli the intracellular
limitations in the bioreaction network for SAP production as the controlling amino
acids. In this case, supply of the controlling amino acids to the fermentation
broth and supplied period are indeed important. In this context, in this study,
first of all the effects of aspartic acid group amino acids, which were reported to
thereafter, the product and by-product distributions of the bioprocess for SAP
production were determined in defined and complex media. Lastly, the results
complex media and to find out the potential strategies in order to increase SAP
and cell yields are much higher in complex medium, than that of defined
medium.
67
The effects of initial aspartic acid concentration on growth and SAP activity were
15mM by using the reference defined production medium. The cell growth and
the cell concentrations obtained at the end of the fermentation were not affected
with the supply of aspartic acid. The highest SAP production was obtained at
0.25 mM aspartic acid concentration with a value of 1074 U cm-3, which was
1.87 fold higher than that obtained for the reference medium. With the further
increase in aspartic acid concentration SAP production decreased slightly but for
The effect of initial asparagine concentration on growth and SAP activity were
obtained as 1082 U cm-3 for an asparagine concentration of 0.25 mM, which was
1.89-fold higher than the reference medium activity. On the other hand, at
cell concentration however; the addition of aspartic acid didn’t affect cell growth.
The effects of initial isoleucine concentration on growth and SAP activity were
for all isoleucine concentrations. The highest SAP activity was obtained as
813 U cm-3 for 0.25 mM concentration, which was 1.4-fold higher than the
reference medium activity. At CIle0 ≥2.5 mM, SAP activity decreased significantly
68
The effects of initial lysine concentration on growth and SAP activity were
bioreactors. Cell growth was slightly inhibited with the addition of lysine. The
highest SAP activity of 926 U cm-3 was obtained at a lysine concentration of 0.25
mM, which was 1.61–fold higher than the reference medium activity. On the
other hand at CLyso≥2.5 mM SAP activity was lower than that obtained for the
reference medium.
The effects of initial threonine concentration on growth and SAP activity were
investigated using r-B.subtilis in the range of 0-15mM. Cell growth and cell
concentrations at the end of the fermentation were higher than that in the
reference medium. At CThr ≥1.0 mM SAP activities were almost the same as the
reference medium. The highest SAP activity was obtained as 900 U cm-3 with an
initial threonine concentration of 0.25 mM which was 1.48-fold higher than the
The effects of initial methionine concentration on growth and SAP activity were
range of 0.25-1.0 mM SAP activity was greater than that obtained for the
reference medium and the highest protease activity was 792 U cm-3 at a
obtained in the reference medium. At CMet≥ 2.5 mM SAP activity were inhibited.
In the study, the product and by-product distributions of the bioprocess for SAP
production were also analyzed in both defined and complex media in order to
medium the glucose consumption rate was higher than in defined medium and
69
The specific glucose consumption rate decreased with the cultivation time both in
defined and complex media. The maximum specific glucose consumption rates in
defined medium and complex medium were 1.97 and 1.42 gG gX-1 h-1,
respectively. The highest cell concentration in defined medium was 1.75 g dm-3
whereas; it was 14.3 g dm-3 in complex medium. Maximum specific growth rates
in defined medium and complex medium were 0.3 and 0.36 h-1, respectively.
The increase in SAP production rate was also higher in complex medium than
that in defined medium. The highest SAP activity obtained in complex medium
was 3–fold higher than defined medium SAP activity as expected, since in order
protease.
In both defined and complex media glycine (12.7 % of the total amino acids in
SAP), serine (11.6 % of the total amino acids in SAP) and valine (11.3 % of the
total amino acids in SAP) were neither observed in the extracellular nor in the
intracellular media indicating that these amino acids were synthesized and
consumed by the cells or synthesized insufficiently. One can also conclude that
the synthesis of these amino acids can be rate-limiting step. Alanine which is
14.5 % of the total amino acids in SAP was not present in the fermentation broth
and was observed only at t=5-10 h in the cell in defined medium. On the other
hand, in complex medium alanine was observed between t=5 h and t=20 h in
the cell and decreased with the cultivation time indicating that alanine present in
the cell at t=5 h was consumed by the cells in the following hours for growth and
in SAP synthesis (Çalık et al., 1999) was not present in the fermentation broth
and was observed only at t=10 h and t=40 h in the cell in defined medium. On
the other hand, in complex medium asparagine was observed in the beginning of
the fermentation and between t=20-45 h in the fermentation broth and t=5 h in
70
the cell. Methionine was observed both in defined and complex media
bioprocess. One can conclude that methionine was supplied from the
fermentation broth and did not synthesized by the cells. On the other hand in
indicating that methionine was synthesized by the cells and did not supplied
from the fermentation broth. Both methionine and asparagine are in the aspartic
acid group amino acids. In the aspartic acid pathway (Figure 2.2) from one
branch asparagine and from another branch methionine are synthesized. The
amino acid analysis results showed that in defined medium asparagine was
neither present in the cell (except t=10 h and t=40 h) nor in the fermentation
broth however, methionine was observed in the cell in high amounts in defined
medium. This is probably due to the lower flux values towards asparagine. The
acid in the extracellular medium indicating that the operation of the TCA cycle
was insufficient. In defined medium lactic acid was observed in the cell
throughout the whole bioprocess whereas in complex medium lactic acid was
the broth also indicates the insufficient operation of the TCA cycle.
71
controlling amino acids. In the study, the effect of supply of aspartic acid group
between 0.25 and 15 mM. The effect of these amino acids on SAP synthesis
medium. The amino acid analysis results showed that serine, valine and glysine
were neither present in the cell nor in the fermentation broth in both defined and
complex media. This may indicate the insufficient synthesis of these amino acids
therefore; the supply of these amino acids to the medium may increase SAP
synthesis, metabolic flux analysis can be done by using the obtained product and
by-product distributions.
72
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77
APPENDIX A
0.8
absorbance @ 600 nm
0.6
0.4
0.2
0
0 0.2 0.4 0.6 0.8
3
CX, kg/m
Absorbance
CX = × Dilution Rate
1.782
78
APPENDIX B
The slope of the calibration curve is m=0.8 1/(µmole cm-3). One unit protease
activity is defined as the activity that liberates 4 nmole of tyrosine per minute.
The activity;
Αbsorbance 1U 1
Α= × Dilution Rate × −1
×
m 4nmole 20min
79
APPENDIX C
0.9
0.8
0.7
0.6
Absorbance
0.5
0.4
0.3
0.2
0.1
0.0
0.0 0.1 0.2 0.3 0.4
CG , kg m-3
Absorbance + 0.2432
CG = × Dilution Rate
3.448
80
APPENDIX D
distilled water.
b) 10 g christalized phenol and 100 cm3 distilled water are added to the
solution.
and mixed.
The solution obtained from the first step is mixed with that from the second
step and then they are stirred until ROCHELLE salt is dissolved. The
after 48 h.
81
APPENDIX E
Place on a magnetic stirrer and stir vigorously. Adjust the pH to 8.0 by adding
approximately 10 g NaOH pellets. The disodium salt of EDTA will not go into
solution until the solution is adjusted approximately pH 8.0. Bring to 500 ml total
volume with dH2O.
Dissolve 60.57 g Tris in dH2O and adjust the pH to 8.0 with concentrated HCl
(approximately 21 ml), cool to room temperature and make the final
adjustments to the pH. Bring to 500 ml with dH2O.
82
APPENDIX F
0.4
0.35
0.3
absorbance @ 540 nm
0.25
0.2
0.15
0.1
0.05
0
0 50 100 150 200 250
CDNA , mg / L
Absorbance
C DNA = × Dilution Rate
0.0018
83
APPENDIX G
0.2
0.18
0.16
absorbance @ 670 nm
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0 50 100 150 200 250
CRNA, m g / L
Absorbance
C RNA = × Dilution Rate
0.0009
84